Electrophoretic polynucleotide fragment analysis methods are used to characterize mixtures of polynucleotide fragments based on their migration velocity through a polymer network under the influence of an electric field, i.e. their electrophoretic mobility, in combination with single or multi-color fluorescence detection. Typically these methods are applied subsequent to amplification of the target polynucleotide using a method such as PCR, e.g. Mullis, U.S. Pat. No. 4,683,202. Examples of such methods include polynucleotide sequencing, e.g. Trainor, Anal. Chem., 62:418-426 (1990), restriction fragment length polymorphisim (RFLP) analysis, e.g. Watkins, Biotechniques, 6:310-319 (1988), and variable number of tandem repeat (VNTR) or microsatellite analysis, e.g. Ziegle et al., Genomics, 14:1026-1031. Each of these methods can provide valuable genetic information about the target polynucleotide.
Current electrophoretic polynucleotide fragment analysis systems are characterized by multiple electrophoresis lanes arranged in a planar array, e.g. a multi-lane slab gel, in combination with a real-time-scanning fluorescence detector, e.g. Hunkapiller et al., U.S. Pat. No. 4,811,218. Multiple lanes are used to increase the overall throughput of the analyzer. In order to collect data during the electrophoresis from multiple lanes, the optical detector system is scanned across the width of the electrophoresis chamber perpendicular to the direction of migration of the labeled polynucleotides. Preferably, multi-color fluorescence detection is used to increase the information density per lane, e.g. for DNA sequencing, four label colors are used, one color for each base. A light source, e.g. a laser, excites the fluorescent labels attached to the polynucleotide fragments, and multiple emission filters discriminate between labels having different spectral properties. In addition, a computer is used to collect data consisting of time, lane number, and fluorescence emission wavelength information, and transform it into useful information, e.g. DNA sequence.
A significant limitation on the speed and resolution of current polynucleotide fragment analysis systems is the ability to dissipate the Joule heat that is generated as a result of the electric current passing through the electrophoresis medium. Because of problems caused by Joule heating, current systems are limited to low, e.g. 25 V/cm, electrical fields, resulting in long analysis times, e.g. 8 hrs. Joule heating and the resulting temperature gradient across the gel can negatively impact the quality of the separation in two ways. First, because heat is generated throughout the electrophoresis medium but only dissipated at its' outside surfaces, a parabolic temperature profile is establish across the depth of the channel. Since electrophoretic velocity is a strong function of temperature, approximately 2% per .degree.C., this temperature profile leads to a parabolic velocity profile for the migrating analytes. This spatial dependence of velocity causes a broadening of the migrating zones, leading to reduced separation performance. The extent of the temperature profile can be reduced by making the electrophoresis channel thinner, e.g. Brumley et al., Nucleic Acids Research, 19:4121-4126 (1991); Stegemann et al., Methods in Molecular and Cellular Biology, 2:182-184 (1991). Therefore, an automated system which incorporates thin electrophoresis channels would be desirable.
Second, if the average temperature of the electrophoresis medium becomes too high, the structural integrity of the medium can be compromised. In the case of polymer gel media, e.g. crosslinked polyacrylamide gels, the elevated temperature can lead to complete destruction of the gel. The average temperature of the electrophoresis medium can be controlled by increasing the rate of heat transfer from the electrophoresis channel to the surrounding environment. Therefore, a system which more efficiently transfers the Joule heat generated as a result of the electrophoresis to the surrounding environment would be desirable.
A further limitation on the speed and resolution of electrophoretic separations is the rate at which the detector can acquire data from fast moving analyte bands. The most desirable form of detection for polynucleotide fragment analysis would be simultaneous multi-color detection. However, current approaches, i.e. an indexable filter wheel in combination with a photomultiplier tube (PMT) detector, are not ideal because the filter wheel must be indexed rapidly enough to observe each color before it moves out of the detector region. This is problematic due to the high electrophoretic velocity of the polynucleotide fragments in high-speed systems. If a sufficient number of data points are not collected for each analyte band, e.g. 10 points per band, the ability to discriminate between adjacent bands is lost. One way to increase the rate of data acquisition for a multi-color system is to collect signals from all the colors simultaneously rather than serially. Therefore, a detection system which acquires all colors simultaneously would be desirable.
In light of the above, what was needed was an improved electrophoresis apparatus capable of accommodating high electric fields through enhanced heat dissipation characteristics and detector performance.